While remarkable advances have occurred in cancer therapy, most available drugs target protein kinases. As a consequence, the number of untapped kinases for new human therapies is dwindling. The Ubiquitin Proteasome System, which degrades unwanted or damaged proteins in the cell, is an attractive new area in drug discovery. Proteins targeted for degradation are covalently modified by a small protein called ubiquitin. The formation of poly-ubiquitin chains onto protein substrates signals the proteaosome that degrades ubiquitylated proteins. Ubiquitin chains are synthesized by two families of enzymes: E2s and E3s. E3s act as a scaffold, recruiting both the protein substrate and the E2. Ubiquitin is transferred from the E2 to the substrate. One area of E3 function that is poorly understood is the mechanism of poly-ubiquitin chain assembly, and gaining a better understanding of how E3s function will be of clear benefit to the drug discovery process. The first step in ubiquitin chain assembly is the transfer of a single ubiquitin to substrate (priming), and the subsequent steps extend the chain (elongation). While it is clear that E2s promote chain elongation, there is still much uncertainty regarding how priming occurs. One thought is that the same E2 responsible for chain elongation also primes the chain. E3s may also recruit separate E2s for priming the substrate and for promoting chain elongation, or the priming step may even involve an E3 rather than an E2. Our hypothesis is that all three of these mechanisms can work in principle, and that the conditions of the reaction determine which model prevails. These include the identities of the E3 and substrate as well as the concentrations of the reaction components. The completion of the specific aims of this grant will identify those conditions. In the first aim, several E3s and their substrates will be assayed in reactions containing either one E2, two E2s, or one E2 and two E3s. The reaction combination that converts the greatest amount of substrate into product will identify the correct model for any given E3-substrate pair. In the second aim, we will test the hypothesis that the presence of two E2s results in slower chain elongation due to competitive inhibition. In the third aim, we will test the hypothesis that E3s are capable of selectively binding to either a priming E2 or E3 and excluding binding to the other. My lab is pioneering the use of pre-steady-state kinetics to measure product formation during the ubiquitylation reaction. This is important because priming and elongation happen so rapidly that it is difficult to distinguish them through more conventional methods.